PLCZmRNA injection: A natural approach to activate bovine somatic cell nuclear transfer embryos

Somatic cell nuclear transfer (SCNT), whereby a somatic cell nucleus is transplanted into an enucleated oocyte, has been successfully used in various mammalian species to derive live cloned offspring. A broad variety of applications that range from agriculture to biomedicine have been envisioned for SCNT; however, its efficiency remains very low. Activation of embryonic development is triggered by the sperm upon fertilization and it involves repetitive increases in intracellular free Ca2+ ([Ca2+]i). Upon fusion with the oocyte, the sperm delivers a factor (phospholipase C-zeta; PLCZ) which induces long-lasting [Ca2+]i oscillations. During SCNT, the activation stimulus must be provided artificially. In cattle SCNT, the most common activation protocols induce a single [Ca2+] i increase and treatment with protein synthesis or kinase inhibitors, which may have detrimental consequences for embryonic development. The hypothesis that an activation protocol that closely mimics the mechanism of activation initiated by the sperm has the potential to improve nuclear reprogramming after SCNT was tested. The activity of bovine- and mouse-derived PLCZ cRNA on bovine oocytes was characterized. Mouse and bovine PLCZ activated development of bovine oocytes in a concentration dependent fashion. At its most effective concentrations, PLCZ induced parthenogenetic development at rates similar to those observed after using common chemical activation protocols. Moreover, PLCZ cRNA injection induced sperm-like [Ca2+]i oscillations and IP3R downregulation. Then PLCZ was implemented as an activation agent for SCNT embryos. SCNT embryos generated by PLCZ cRNA injection where compared with those produced by standard chemical activation protocols (lonomycin/DMAP and lonomycin/CHX), and to IVF-derived embryos as controls. PLCZ cRNA injection induced [Ca2+]i oscillations similar to those observed after fertilization and induced blastocyst development at rates similar to IVF embryos. Gene expression analysis at 8-cell and blastocyst stages revealed that some abnormalities observed in SCNT embryos activated by chemical means were not found in SCNT embryos activated using PLCZ cRNA injection. Finally, chromatin modifications were analyzed at the blastocyst stage. The levels of trimethylated lysine 27 at histone H3 were higher in bovine SCNT embryos activated using CHX and DMAP than in those activated using PLCZ or derived from IVF. In summary, this dissertation shows that PLCZ cRNA injection can; (1) induce sperm-like calcium oscillations, (2) be effectively used to trigger the initiation of development in SCNT embryos, and (3) produce cloned embryos that have more similar characteristics to their IVF counterpart than chemically activated cloned embryos.